Mechanisms of release and signaling functions of ciliary vesicles

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In the mammalian male reproductive system, extracellular vesicles released by somatic epithelial cells participate to the maturation of spermatozoa through the transfer of molecular cargos. This unique process of soma-to-sperm communication occurs in the tubule attached to the testis, named the epididymis, and supports the acquisition of sperm motility and fertilizing abilities. Despite the important role played by extracellular vesicles to sustain male reproductive functions, their cellular source, mechanisms of release and respective signaling functions remain largely unknown. Primary cilia are solitary sensory antennae that protrude upward from the cell surface to form a subcellular compartment enriched in signaling molecules. Vesicles derived from these organelles have been shown to contain a specific subset of bioactive molecules. We recently unveiled that primary cilia are abundant in the tubules located upstream of the epididymis, i.e. in the efferent ductules. In this location, they release extracellular vesicles that express specific ciliary markers (e.g Arl13b and Cytochrome p450 oxidoreductase (POR)) into the epididymal fluid. With the hypothesis that ciliary extracellular vesicles from the epididymis may constitute news means of communication between ciliated somatic cells and the maturing spermatozoa, we propose a research program articulated around 3 majors aims: Aim 1. Characterize ciliary vesicles from the male reproductive system We will take advantage of a double transgenic mouse model, in which the Arl13b ciliary marker is fused with m-cherry tag, to detect and sort primary cilia-deriving extracellular vesicles by dedicated small particle flow-cytometry approaches (collaboration E. Boilard, U.Laval). The size, protein markers and molecular content of m-cherry positive ciliary vesicles will be determined and compared with m-cherry negative extracellular vesicles from the epididymis. Aim 2. Determine their mechanisms of release Vesicles that derive from the primary cilium of endothelial cells are formed by outward budding of the plasma membrane under the control of shear stress and intraflagellar transport (IFT) components. We will test the contribution of these two factors on epithelial primary cell cultures isolated from the efferent ductules of wild type and IFT88 knock-out mice by using biofludic imaging system. Aim 3. Assess their ability to modulate sperm content and function Since extracellular vesicles participate to intercellular communication via the transfer of bioactive molecules, we will assess their role in somatic cells-spermatozoa communication by combining in vitro co-incubation assays with in vivo functional studies on knock-out mouse models. This NSERC research will unravel the unique features and functions of ciliary vesicles in the soma-to-sperm communication system and may identify new cellular mechanisms that control sperm maturation and male reproductive functions.